It is thought that fucose-containing sugar chains are not only associated with such vital phenomena as development, differentiation and cell recognition, but are also associated with the onset and progression of inflammations, cancers, autoimmune diseases and many other diseases (Edited by Akira KOHATA, Senichiro HAKOMORI, Katsutaka NAGAI, Glycobiology Series 1–6, Kodansya, (1993), Glycobiology, 3, 97 (1993)).
Fucose-containing sugar chains are bound to proteins or lipids, and thus exist not only in the form of glycoproteins, proteoglycans and glycolipids, but also in the form of oligosaccharides.
The Lewis x sugar chain [Galβ1-4(Fucα1-3)GlcNAc], one of the fucose-containing sugar chains, appears regularly in each developmental stage of the early embryo, brain and kidney. For the reasons that the Lewis x sugar chains have affinity with one another and that they are associated with the cell compaction which occurs in the developmental stage of mouse early embryos, it is considered that the Lewis x sugar chain plays an important role in the development of early embryo, brain and kidney, acting as an adhesion molecule or a sugar chain signal.
For this reason, by cloning a gene of α1,3-fucosyltransferase involved in the synthesis of the Lewis x sugar chain appearing in the developmental stage of those organs, and analyzing its role, it is possible to clarify the relation between the gene and hereditary diseases.
The sialyl Lewis x sugar chain [NeuAcα2-3Galβ1 1-4(Fucα1-3)GlcNAc], the sialyl Lewis a sugar chain [NeuAcα2-3Galβ1-3(Fucα1-4)GlcNAc], the Lewis x sugar chain and the Lewis y sugar chain [Fucα1-2Galβ1-4(Fucα1-3)GlcNAc] are known as cancer-associated sugar chains.
The sialyl Lewis a sugar chain is detected mainly in cancers developing in the digestive system such as colon cancer and pancreatic cancer with high frequency. On the other hand, the sialyl Lewis x sugar chain, the Lewis x sugar chain and the Lewis y sugar chain are detected in lung cancer, ovarian cancer and renal cancer as well as the cancers developing in the digestive system.
Since it is clear that, even if the same sugar chain appears, glycosyltransferase involved in the synthesis of the sugar chain varies depending on the cell, it is considered possible to diagnose cancers more correctly, for example, by identifying α1,3-fucosyltransferase involved in the biosynthesis of the Lewis x sugar chains in each cancer cell and clarifying the correlation between the transferase and the specific cancer.
The Lewis x sugar chain is also involved in both the clearance in the blood and targeting to the organ of interest in respect of the glycoprotein containing the sugar chain. It is considered possible to control the clearance rate of any protein in the blood or to target to the liver by artificially adding the Lewis x sugar chain to any protein (J. Biol. Chem., 270, 24024–24031 (1995)).
Furthermore, since the Lewis x sugar chain is known to have an activity to adhere reciprocally among the sugar chains (Glycoconjugate J., 11, 238–248 (1994), Glycobilogy, 8,139–146 (1998)), after adding the Lewis x sugar chain to any protein, it is also possible to make the protein target to a cancer organ which highly expresses the Lewis x sugar chain.
In the case where the sialyl Lewis x sugar chain is added to a protein, it is thought that the protein targets a cell expressing selectin (e.g. vascular endothelial cell in inflammatory site).
Hence, in order to make any protein target to the liver or cancer tissues effectively, it is desirable that the Lewis x sugar chain is effectively added to the protein, while the sialyl Lewis x sugar chain is not added thereto. It is an industrially important object to develop a method for effectively adding the Lewis x sugar chain to any recombinant glycoprotein, without adding the sialyl Lewis x sugar chain thereto, in a host cell suitable for producing the recombinant glycoprotein (e.g. Namalwa cell, Namalwa KJM-1 cell, CHO cell etc.)
It has been reported that, when an α1,3-fucosyltransferase (Fuc-TIV) is expressed in a specific CHO cell line, the Lewis x sugar chain is expressed without expression of sialyl Lewis x sugar chain (J. Biol. Chem., 266, 17467–17477(1991), J. Biol. Chem., 266, 21777–21783(1991)). However, since Fuc-TIV originally has an activity to synthesize the sialyl Lewis x sugar chain, when Fuc-TIV is expressed in other cells (e.g. other types of CHO cell lines or Namalwa KJM-1 cell), the sialyl Lewis x sugar chain is expressed in addition to the Lewis x sugar chain (Cell., 63, 1349–1356, J. Biol. Chem., 269, 14730–14737(1994)).
Some reasons why the amount of the sialyl Lewis x sugar chain synthesized by Fuc-TIV is different depending on the cells into which Fuc-TIV is introduced may be that the amount and kind of sugar chain acting as a substrate in these cells is different, and that the expression level of the expressed Fuc-TIV or other glycosyltransferases (e.g. α2,3-siaryltransferase) is different among these cells.
It is thought that, for example, α2,3-siaryltransferase (ST3Gal III or ST3Gal IV) and Fuc-TIV compete for the same substrate (a sugar chain having an N-acetyllactosamine structure).
When the activity of α2,3-siaryltransferase is relatively stronger than that of Fuc-TIV, an α2,3-sialyl-N-acetyllactosamine sugar chain is generated with higher priority, and then fucose is added to the sugar chain by Fuc-TIV to synthesize the sialyl Lewis x sugar chain. In contrast, when the activity of Fuc-TIV is relatively stronger than that of α2,3-siaryltransferase, fucosyl-N-acetyllactosamine sugar chain (the Lewis x sugar chain) is synthesized with higher priority. Since α2,3-siaryltransferase has low activity to add sialic acid to the Lewis x sugar chain, it is difficult to synthesize sialyl Lewis x sugar chain with α2,3-siaryltransferase.
Thus, it is considered that, if a novel α1,3-fucosyltransferase, which has an activity to synthesize the Lewis x sugar chain but does not have an activity to synthesize the sialyl Lewis x sugar chain, can be obtained, it is possible to effectively synthesize the Lewis x sugar chain without synthesizing the sialyl Lewis x sugar chain by expressing the transferase in a host cell. Many cells including cells suitable for substance production have N-acetyllactosamine structure which is a precursor of the Lewis x sugar chain. Accordingly, the isolation of the above-stated novel enzyme will make it possible to effectively synthesize the Lewis x sugar chain by using various kinds of cells as host cells, without synthesizing the sialyl Lewis x sugar chain. However, such an enzyme is still unknown.
Since anti-CD15 antibody which recognizes the Lewis x sugar chain binds to human sperm after acrosome reaction and inhibits the interaction between the sperm and an egg, it is assumed that the Lewis x sugar chain is deeply involved in fertilization (American Journal of Reproductive Immunology, 37, 172–183 (1997)). So, for the diagnosis and treatment of infertility disease, it is useful to identify an a α1,3-fucosyltransferase involved in the biosynthesis of the Lewis x sugar chain existing in a human sperm and clarify the relation between the transferase and infertility desease. Also, there is a possibility of establishing a safe and reliable contraception method, using a protein to which the Lewis x sugar chain or the Lewis y sugar chain is added.
Therefore, for these purposes also, the development of a method for effectively adding the Lewis x sugar chain to any protein is very important.
It has been found that the binding between selectins (E-, P- and L-selectin), an adhesive molecules and their sugar chain ligand (the sialyl Lewis x sugar chain or its relative sugar chains) is involved in the accumulation of leukocytes into inflammatory sites or homing of lymphocytes to a lymph nodes.
The sialyl Lewis x sugar chain and its stereo isomer, the sialyl Lewis a sugar chain are known as cancer-associated antigens whose expression level increases with canceration, and the antibodies recognizing these sugar chains are used for the serodiagnosis of cancers.
Since these sugar chains bind to selecting, it is assumed that these sugar chains are also involved in the metastasis of cancers. Recently, it has been discovered that glycolipid containing the Lewis x sugar chains existing in mouse kidney strongly bind to E-selectin (Biochem. Biophys. Res. Commun., 218, 610–615 (1996)).
A sugar chain having a strong binding acitivity to selectins is useful for the treatment and prevention of inflammation and metastasis of cancers, acting as a selectin antagonist. α1,3-fucosyltransferases expressed in a mouse kidney are assumed to be useful in the effective synthesis of selectin antagonists, and identification and production of the above enzymes are considered to be significant. Nevertheless, the above enzymes have not been identified yet.
To date, genes for 5 types of α1,3-fucosyltransferases (Fuc-TIII, Fuc-TIV, Fuc-TV, Fuc-TVI and Fuc-TVII) have been cloned, and acceptor substrate specifity of each enzyme has been analyzed (Kukowska-Latallo et al., Genes &Dev. 4, 1288–1303 (1990), Goelz et al., Cell 63, 1349–1356(1990), Lowe et al., J. Biol. Chem. 266, 17467–17477(1991), Kumar et al., J. Biol. Chem. 266, 21777–21783(1991), Weston et al., J. Biol. Chem. 267, 4152–4160(1992), Weston et al., J. Biol. Chem. 267, 24575–24584(1992), Koszdin et al., Biochem. Biophys. Res. Commun. 187, 152–157(1992), Sasaki et al., J. Biol. Chem. 269, 14730–14737(1994), Natsuka et al., J. Biol. Chem. 269, 16789–16794(1994)).
Fuc-TIII has both α1,3-fucosyltransferase activity and α1,4-fucosyltransferase activity and is capable of synthesizing the sialyl Lewis x sugar chain, the Lewis x sugar chain, the Lewis y sugar chain, the sialyl Lewis a sugar chain, the Lewis a sugar chain [Galβ1-3 (Fucα1-4) GlcNAc] and the Lewis b sugar chain [Fuc α1-2Galβ1-3 (Fucα1-4) GlcNAc] (Fuc-TIII has a strong acitivity to synthesize the Lewis a sugar chain and Lewis b sugar chains).
Fuc-TIV is capable of synthesizing the sialyl Lewis x sugar chain, the Lewis x sugar chain and the Lewis y sugar chain (Fuc-TIV has a strong activity to synthesize the Lewis x sugar chain and the Lewis y sugar chain).
Fuc-TV is capable of synthesizing the sialyl Lewis x sugar chain, the Lewis x sugar chain and the Lewis y sugar chain.
Fuc-TVI is capable of synthesizing the sialyl Lewis x sugar chain, the Lewis x sugar chain and the Lewis y sugar chain.
Fuc-TVII is capable of synthesizing only the sialyl Lewis x sugar chain.
As described above, each of the cloned 5 types of α1,3-fucosyltransferases seems to have a somewhat-similar acceptor substrate specificity, but in the strict sense, each enzyme has a distinct acceptor substrate specificity. Furthermore, it is clear that the expression of each enzyme is specific for a cell or stage and that multiple enzymes may be expressed in one cell simultaneously. Even though the above enzymes can synthesize fucose-containing sugar chains having similar structure, it is thought that respective enzyme has a distinct function, because of the difference in cells or stages of their expression.
By an enzymological analysis using the extracts from cells or tissues as an enzyme source, it is possible, to a certain extent, to identify an α1,3-fucosyltransferase which expresses in a tissue or cell (Mollicone et al., Carbohydrate Research, 228, 265–276 (1992), Weston et al., J. Biol. Chem., 267, 24575–24584 (1992)). However, with the enzymological analysis, it is impossible to identify respective α1,3-fucosyltransferases expressed in a cell or tissue expressing multiple α1,3-fucosyltransferase, and to clarify the enzymologic characteristic of each α1,3-fucosyltransferase.
In order to detect the expression of a specific α1,3-fucosyltransferase, it is necessary to use an immunological detection method using a specific antibody or a detection method based on the nucleotide sequence of genes (e.g. Northern hybridization or PCR).